Original Paper Int Arch Allergy Immunol 2013;160:221–232 DOI: 10.1159/000339740
Received: July 4, 2011 Accepted after revision: May 24, 2012 Published online: October 16, 2012
Structural and Immunological Characterization of Recombinant Pan b 1, a Major Allergen of Northern Shrimp, Pandalus borealis Heidi R. Myrset a Bianca Barletta b Gabriella Di Felice b Eliann Egaas a Maaike M.B.W. Dooper a a Norwegian
Veterinary Institute, Oslo, Norway; b Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy
Key Words Recombinant allergens ⴢ Tropomyosin ⴢ Shrimp ⴢ Allergens ⴢ Food allergy ⴢ IgE ⴢ Circular dichroism ⴢ Mass spectroscopy ⴢ Basophil activation test ⴢ Skin prick test
Abstract Background: Shellfish allergy is one of the major causes of life-threatening allergic reactions to food. The shrimp species Pandalus borealis is the commercially most important coldwater shrimp species, and its protein extract is commonly used in shrimp allergy diagnostics. However, the DNA sequence of its major allergen, tropomyosin, designated Pan b 1, was not previously described. Our aim was to identify the cDNA sequence of Pan b 1 and to generate a recombinant protein with similar structure and allergenicity as the natural protein. Methods: P. borealis shrimps were caught in the Oslofjord (Norway). cDNA from Pan b 1 was generated, an Nterminal histidine tag was added, and the protein was expressed in Escherichia coli. The recombinant Pan b 1 was characterized by structural and IgE-binding studies and investigated further with basophil activation tests (BATs) and skin prick tests (SPTs) on Norwegian shrimp-allergic individuals. Results: The open reading frame encoded 284 amino acids that shared 97–100% identity with other shrimp tropomyosins. Mass spectroscopy of natural Pan b 1 confirmed the
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protein’s molecular mass and indicated the absence of posttranslational modifications. Circular dichroism spectroscopy revealed virtually identical spectra between recombinant and natural Pan b 1, which together with native PAGE and size exclusion chromatography results indicated a similar structure. Furthermore, immunoblot and ELISA studies as well as BATs and SPTs showed equivalent results of recombinant and natural Pan b 1. Conclusion: A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy. Copyright © 2012 S. Karger AG, Basel
Introduction
Shellfish allergy is a potentially life-threatening disease that is seldom outgrown [1, 2] and, in some parts of the world, the most common food allergy among adults [3]. The shellfish species that most frequently elicit foodallergic reactions belong to the taxonomic class Crustacea, which includes shrimp, crab, crawfish and lobster. Affected individuals usually display allergic reactivity to multiple crustacean species. Molecular and clinical crossreactivity was reported between crustaceans and other invertebrate foods such as mussels, oyster, squid and ocCorrespondence to: Dr. Heidi Ragnhild Myrset Norwegian Veterinary Institute PO Box 750 Sentrum NO–0106 Oslo (Norway) Tel. +47 2321 6263, E-Mail heidi.myrset @ gmail.com
topus, but also invertebrate aeroallergens such as house dust mite and cockroaches (reviewed by Lopata et al. [1]). The presence of a heat-stable allergen in shellfish was first identified in shrimp by Hoffman et al. [4], and this allergen was later identified as the muscle protein tropomyosin [5–7]. More than 80% of shrimp-allergic individuals were reported to have serum IgE against shrimp tropomyosin [6, 8–10]. Shrimp tropomyosin also inhibited 75% of IgE binding to whole-shrimp extract in radioallergosorbent tests [6]. The amino acid sequence of invertebrate tropomyosins is highly conserved, with 95% identity between shrimp and storage mite (Tyrophagus putrescentiae). Tropomyosin was found to play an important role in the cross-reactivity seen between the different invertebrate species, suggesting tropomyosin to be an invertebrate pan-allergen [1]. Tropomyosin constitutes 20% of the water-soluble protein of shrimp meat [11] and plays a role in the contractile activity of muscle cells [12]. It is present as a homodimer of two ␣-helical molecules that are generally 284 amino acids long and wound around each other to form a coiled-coil structure of approximately 40 nm in length [12]. Shrimp tropomyosin migrates as a 34- to 39kDa band in SDS-PAGE [1], although the calculated molecular mass of the amino acids is 33 kDa. Muscle tropomyosin has been described as a water-soluble protein containing an N-terminal acetylation [12] that might be critical for the polymerization and function of tropomyosin [13]. Shrimp tropomyosin was also reported to contain a small percentage of carbohydrates [4, 6], and a recent study suggested that mature shrimp tropomyosins are formed by the elimination of a leader peptide of 9 amino acids [14]. Shrimp tropomyosin was first cloned from the sand shrimp Metapenaeus ensis (Met e 1) by Leung et al. [15] and afterwards from several other shrimp species such as the brown shrimp Farfantepenaeus aztecus (Pen a 1) [16] and the Alaskan pink shrimp Pandalus eous [17]. Recombinant allergens are considered useful for allergen standardization, improvement of diagnostics and the development of novel strategies of allergen-specific immunotherapy (reviewed by Valenta et al. [18]). The Northern Atlantic shrimp Pandalus borealis is the commercially most important coldwater shrimp species [19] and the shrimp species most commonly eaten in Norway. Furthermore, it is frequently used in shrimp extracts for shrimp allergy diagnostics [Phadia ImmunoCap and ALK-Abelló skin prick test (SPT) extract]. However, the sequence of tropomyosin from P. borealis has not yet been described. The aim of the present study was therefore to 222
Int Arch Allergy Immunol 2013;160:221–232
identify the cDNA sequence of tropomyosin from P. borealis and to generate a recombinant form of the protein that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.
Materials and Methods Bioinformatics Amino acid alignment and sequence identities were obtained using ClustalW and Boxshade (http://mobyle.pasteur.fr/cgi-bin/ portal.py?#forms::boxshade) with data from GenBank. cDNA Synthesis and Cloning P. borealis shrimps were caught in the Oslofjord (Norway). The tips of the tails from live P. borealis were cut off and stored at 4 ° C in RNAlater Stabilization Reagent (Qiagen, Hilden, Germany) until homogenization (MagNALyzer, Roche Diagnostics, Mannheim, Germany) and isolation of total RNA (RNeasy Fibrous Tissue Mini Kit, Qiagen). cDNA was obtained by RT-PCR with random hexamer primers (SuperScriptTM III First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, Calif., USA). Tropomyosin cDNA was amplified with gene-specific primers deduced from the untranslated regions of P. eous tropomyosin mRNA (GenBank accession No. AB270631; forward primer, 5ⴕCTTTCTCAGTTCTTTTTGTTGG-3ⴕ and reverse primer, 5ⴕAAAGTGAATAATGAGATAAAATAAGC-3ⴕ). The PCR parameters were as follows: 95 ° C for 1 min, 40 cycles of 95 ° C for 20 s, 50 ° C for 20 s and 72 ° C for 32 s, and finally 72 ° C for 3 min. The PCR product was sequenced by GATC Biotech (Konstanz, Germany) with the same primers. The identified isoform of P. borealis tropomyosin was designated Pan b 1.0101. The open reading frame of the cDNA was amplified and cloned into the vector pET19b (Novagen, Darmstadt, Germany) using In-Fusion cloning (Clontech, Saint-Germain-en-Laye, France) with 15 overlapping base pairs from the vector, according to the instructions from the manufacturer. The following primers were used: forward primer, 5ⴕ-GACGACGACGACAAGATGGA CGCCATCAAGAAGAAG-3ⴕ and reverse primer, 5ⴕ-GCTTTGT TAGCAGCCTTAGTAGCCAGACAGTTCGCTGA-3ⴕ (the overlapping base pairs are underlined). The cDNA clone was expanded in XL10-Gold cells (Stratagene, San Diego, Calif., USA), plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen) and the insert was sequenced by GATC Biotech. The pET19b vector encoding Pan b 1 connected to an N-terminal decahistidine tag and an enterokinase cleavage site (MGHHHHHHHHHHSSGHIDDDDK) was then transformed into Escherichia coli RosettaTM 2(DE3) competent cells (Novagen) for expression. The recombinant His-tagged form of Pan b 1.0101 is referred to as rPan b 1 in the text of this article. All primers were synthesized at Eurofins MWG (Ebersberg, Germany).
Expression and Purification of rPan b 1 Expression of rPan b 1 was performed using the Overnight ExpressTM Autoinduction System 1 (Novagen). Cells were harvested by centrifugation (1 h, 5,500 g, 4 ° C) and stored at –80 ° C overnight. Protein was extracted using 5 ml/g of pellet of a denaturing extraction buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imid
Myrset /Barletta /Di Felice /Egaas /Dooper
azole, 8 M urea, pH 7.4). rPan b 1 was purified by immobilized metal affinity chromatography (IMAC) using HisPur Cobalt Spin Columns according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, Ill., USA). Final purification and buffer exchange to 3-(N-morpholino)propanesulfonic acid (MOPS; 20 mM, 500 mM NaCl, pH 7.4) was performed by size exclusion chromatography [SEC; Superdex 75 pg (16/60), GE Healthcare, UK]. IMAC-purified rPan b 1 was used in the protein identification by mass spectroscopy (MS), while for all the other studies the samples were also SEC-purified. Protein concentrations were determined according to the Pierce 660 method (Pierce Biotechnology) for samples containing 8 M urea and according to the Lowry method for all other samples (DC protein assay, Bio-Rad, Hercules, Calif., USA) using bovine serum albumin (BSA) as a standard. His-tag Removal The decahistidine tag and the additional amino acids of the enterokinase cleavage site were removed by enterokinase cleavage using 1 U per 40 g of protein for 16 h at room temperature (Enterokinase Cleavage Capture Kit, Novagen). Identification of rPan b 1 by MS rPan b 1 was purified with IMAC and SDS-PAGE, and the 39kDa protein was excised from the Coomassie Blue-stained gel. The protein was digested by 3 l of trypsin (porcine trypsin, Promega, USA; 100 ng/l dissolved in 50 mM acetic acid) at a pH of 8 for 16 h at 37 ° C. The peptides were analyzed on an Ultraflex III matrix-assisted laser desorption ionization (MALDI)-time-offlight (TOF)/TOF mass spectrometer (Bruker Daltronics GmbH, Bremen, Germany) operated in reflector mode and used at an accelerating potential of 25 kV (at the Proteomics Unit at the University of Bergen, Norway). A number of MS spectra, each generated with 200 shots, were accumulated. The peptide mapping data of the protein were analyzed using the Mascot search engine (http://www.matrixscience.com/), with NCBI as database and taxonomy metazoa chosen as parameters.
(Millipore, Billerica, Mass., USA) and directly eluted onto a stainless steel sample holder with 0.6 l of matrix solution [20 mg/ml ␣-cyano-4-hydroxycinnamic acid in 0.3% aqueous trifluoroacetic acid/acetonitrile (2:1)]. After recording the MS spectra, 0.4 l of horse cytochrome C (1 pmol/l) was added to the rPan b 1 sample and 0.4 l of BSA (1 pmol/l) was added to the natural Pan b 1 sample for internal calibration. MS spectra were transformed into peak lists using the software FlexAnalysis version 2.4 (Bruker Daltonics). The basic settings of the MALDI-TOF/TOF instrument (Ultraflex II, Bruker Daltonics) were as follows: ion source 1, 25 kV; ion source 2, 23.8 kV; lens, 6.80 kV; gating mode with high gating strength; pulsed ion extraction, 140 ns; polarity positive, and suppression up to 7,000 Da. Circular Dichroism Spectroscopy Recombinant and natural Pan b 1 were dialyzed against 10 mM KH2PO4/K 2HPO4 buffer (pH 7.4), and the protein concentrations were adjusted to approximately 125 g/ml. Circular dichroism spectra were recorded and analyzed by Dr. Kristin Gunnarsen (Centre for Immune Regulation, University of Oslo, Norway) as previously described [23]. Study Subjects Six individuals with positive SPTs to shrimp extract (P. borealis, ALK-Abelló A/S, Hoersholm, Denmark) were recruited at the Haukeland University Hospital (Bergen, Norway). For control experiments, 6 individuals with negative SPTs to shrimp extract were included for the basophil activation tests (BATs), and 3 nonatopic individuals were included for the SPTs. Serum samples were stored at –20 ° C until further analyses. The clinical and laboratory features of the shrimp-allergic individuals are listed in table 1. Approval of the studies involving human subjects was obtained from the Norwegian National Ethical Board.
Whole-Shrimp Extract and Natural Pan b 1 Whole-shrimp extract and natural Pan b 1 were obtained from frozen, peeled, boiled P. borealis caught in the Oslofjord (Norway). Whole-shrimp extract was prepared essentially according to the methods reported in the literature with some modifications [20, 21]. Whole shrimp bodies were blended for 3 min in 0.01 M (1:10 w/v) phosphate-buffered saline (PBS) and boiled for 15 min. After stirring at 4 ° C for 5 h, the proteins were dialyzed for 48 h against several changes of bidistilled water using a 10,000 molecular weight cutoff membrane (Spectrum Laboratories Inc., Rancho Dominguez, Calif., USA) and freeze dried. Natural Pan b 1 was extracted as previously described [22], followed by SEC purification as described for rPan b 1. The protein concentration of whole-shrimp extract and natural Pan b 1 was determined by the Lowry method.
SDS-PAGE and Immunoblotting Proteins were separated by SDS-PAGE under reducing conditions using 4–12% Bis-Tris precast gels (Invitrogen) and detected by SimplyBlue SafeStain (Invitrogen). For immunoblot analysis, proteins were electrophoretically transferred from the gels to nitrocellulose membranes (pore size 0.45 m, Bio-Rad). Membranes were blocked with PBS containing 0.05% Tween-20 (PBST) and 3% horse serum for 1 h, following incubation with patient serum (diluted 1:30) overnight at 4 ° C in blocking buffer. For IgE detection, membranes were first incubated with rabbit antihuman IgE (1: 6,000; DakoCytomation, Glostrup, Denmark) and then with goat antirabbit IgG horseradish peroxidase conjugate (1: 5,000; Zymed, San Francisco, Calif., USA), each for 1 h. IgE binding was revealed with 3,3ⴕ 5,5ⴕ-tetramethylbenzidine substrate (Single solution, Zymed). In between each incubation, blots were washed three times with PBST for 15 min. All incubation and washing steps were performed at room temperature with gentle shaking. Novex쏐 Sharp Pre-Stained Protein Standard (Invitrogen) was used as the protein size marker.
Molecular Mass Determination of Recombinant and Natural Pan b 1 by MS Recombinant and natural Pan b 1 were analyzed in the linear TOF mode of an Ultraflex II (Bruker Daltonics) MALDI-TOF/ TOF mass spectrometer (at the Biotechnology Centre of Oslo, Norway). The protein samples were purified using a ZipTipC4
Native PAGE SEC-purified proteins and protein extracts were separated by SDS-PAGE under nonreducing conditions using native 10% TrisGlycine precast gels (Invitrogen) and were detected by SimplyBlue SafeStain (Invitrogen). NativeMark Unstained Protein Standard (Invitrogen) was used as the protein size marker.
Immunological Characterization of Recombinant Pan b 1
Int Arch Allergy Immunol 2013;160:221–232
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Table 1. Clinical and laboratory features of shrimp-allergic individuals
Donor
Age Sex Dominating years symptoms
Total IgE sIgE shrimp kU/l kUA/l
sIgE rPen a 1 kUA/l
SPT shrimp mm
SPT nPan b 1 mm
Positive SPT to other invertebrates
A B C D E F
27 49 37 38 33 37
328 71 780 51 124 242
39.9 4.02 5.07
